Lung cancer is the leading cause of cancer mortality worldwide with lung adenocarcinomas representing the subtype that is increasing in incidence. Identification of specific alterations such as EGFR mutations and more recently, ALK kinase gene fusions, have lead to the more efficacious treatment of patients whose lung adenocarcinomas contain these alterations. We have utilized a novel strategy for the identification of gene fusions in lung cancer (Wang et al, Nature Biotechnology) revealing the R3HDM2-NFE2 and more recently the HSPA1A-NFKBIL1 gene fusions in lung adenocarcinomas, as well as nominating many additional potential gene-fusion candidates. We hypothesize that identification of novel gene fusions in lung adenocarcinoma may provide new avenues for highly selective treatments of this cancer as well as specific markers for cancer detection or therapeutic monitoring. Three specific aims are proposed. Aim one is to functionally characterize the novel R3HDM2-NFE2 gene fusion utilizing siRNA knockdown in lung cancer lines containing these alterations as well as using lung cell lines engineered to overexpress NFE2. Effects on tumor cell proliferation, invasion and specific transcriptional programs will define the mechanisms underlying R3HDM2-NFE2 oncogenic activity. Aim two is to define the frequency of occurrence of the novel HSPA1A-NFKBIL1 gene fusion in primary lung cancers using fluorescence in situ hybridization with tissue microarrays, followed by functional analyses of the oncogenic activity of the fusion gene as those utilized for the R3HDM2-NFE2 in Aim one. Aim three will employ the successful methodologies used for the discovery of the R3HDM2-NFE2 and HSPA1A- NFKBIL1 fusions, to identify, validate and functionally characterize additional novel fusions involving functionally important cellular processes in lung cancer. Priority will be gene fusions that are candidates for targeted therapeutics or that involve genes and critical cellular targets for which therapies may be potentially developed. Higher emphasis will also be placed on candidates occurring at high frequency or within identifiable tumor subgroups. These studies have potential to identify new approaches to treat the leading cause of cancer death.